The aim of this study was to report the use of novel masking agents during an anterior lamellar keratoplasty performed using a femtosecond laser in a patient with corneal ectasia that was consistent with recurrent keratoconus.

All organisms react to noxious and mechanical stimuli but we still lack a complete understanding of cellular and molecular mechanisms by which somatosensory information is transformed into appropriate motor outputs. The small number of neurons and excellent genetic tools make Drosophila larva an especially tractable model system in which to address this problem. We developed high throughput assays with which we can simultaneously expose more than 1,000 larvae per man-hour to precisely timed noxious heat, vibration, air current, or optogenetic stimuli. Using this hardware in combination with custom software we characterized larval reactions to somatosensory stimuli in far greater detail than possible previously. Each stimulus evoked a distinctive escape strategy that consisted of multiple actions. The escape strategy was context-dependent. Using our system we confirmed that the nociceptive class IV multidendritic neurons were involved in the reactions to noxious heat. Chordotonal (ch) neurons were necessary for normal modulation of head casting, crawling and hunching, in response to mechanical stimuli. Consistent with this we observed increases in calcium transients in response to vibration in ch neurons. Optogenetic activation of ch neurons was sufficient to evoke head casting and crawling. These studies significantly increase our understanding of the functional roles of larval ch neurons. More generally, our system and the detailed description of wild type reactions to somatosensory stimuli provide a basis for systematic identification of neurons and genes underlying these behaviors.

The systemic lupus erythematosus (Sle1) interval from the NZM2410 mouse strain has been shown to be responsible for high levels of autoantibody production against antinuclear antibodies (ANA) when transferred into C57BL/6 mice. B cells derived from the B6.Sle1 strain are required for the production but help from both T-dependent and independent sources have been documented. Using radiation chimeras constructed in a strain of mice that is chronically depleted of Natural killer (NK) cells, but not NKT cells, we have examined the role of NK cells in the development of ANA in this context. Our results show that in the presence of intact T cell help depletion of NK cells does not affect ANA production. However, when T cell help is compromised, the prevalence of animals producing ANA is significantly decreased suggesting that NK cells can provide help for the T-independent production of ANA. Further experiments provide a possible mechanism for the NK-cell dependence.

Clostridium difficile, a leading cause of hospital-acquired bacterial infection, is coated in a dense surface layer (S-layer) that is thought to provide both physicochemical protection and a scaffold for host-pathogen interactions. The key structural components of the S-layer are two proteins derived from a polypeptide precursor, SlpA, via proteolytic cleavage by the protease Cwp84. Here, we report the design, synthesis and in vivo characterization of a panel of protease inhibitors and activity-based probes (ABPs) designed to target S-layer processing in live C. difficile cells. Inhibitors based on substrate-mimetic peptides bearing a C-terminal Michael acceptor warhead were found to be promising candidates for further development.

The development and application of chemical technologies enabling direct analysis of enzyme activity in living systems has undergone explosive growth in recent years. Activity-based protein profiling (ABPP) is a key constituent of this broad field, and is among the most powerful and mature chemical proteomic technologies. This tutorial review introduces the essential features of ABPP and the design and application of activity-based probes (ABPs) from drug target elucidation and in vivo visualisation of enzyme activity to comprehensive profiling of the catalytic content of living systems, and the discovery of new biological pathways.

Clostridium difficile, a leading cause of hospital-acquired infection, possesses a dense surface layer (S-layer) that mediates host-pathogen interactions. The key structural components of the S-layer result from proteolytic cleavage of a precursor protein, SlpA, into high- and low-molecular-weight components. Here we report the discovery and optimization of the first inhibitors of this process in live bacteria and their application for probing S-layer processing. We also describe the design and in vivo application of activity-based probes that identify the protein Cwp84 as the cysteine protease that mediates SlpA cleavage. This work provides novel chemical tools for the analysis of S-layer biogenesis and for the potential identification of novel drug targets within clostridia and related bacterial pathogens.

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